Methods of preparing a polymeric latex composition and water-insoluble biological reagent

ABSTRACT

A polymeric latex composition having a hydrophobe incorporated therein can be prepared using a novel latex &#34;loading&#34; method. This method includes providing a loadable latex, providing a hydrophobe dissolved in a water-miscible organic solvent, heating the loadable latex to about 30° to about 90° C. and gradually adding the hydrophobe solution to the heated loadable latex under conditions to keep the hydrophobe in solution and to &#34;load&#34; it into the latex particles. In preferred embodiments, the latex is surfactant-free and has particles which have outer reactive groups which are capable of reacting with free amine or sulfhydryl groups of a biological compound. Such latices can be used to prepare water-insoluble biological reagents having a biological compound attached to the particles.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.07/864,885, filed Apr. 3, 1992 now issued as U.S. Pat. No. 5,234,841,which in turn is a continuation of U.S. application Ser. No. 07/612,364filed Nov. 14, 1990, now abandoned, which in turn is a divisional ofU.S. application Ser. No. 347,537 filed May 4, 1981, now issued as U.S.Pat. No. 5,053,443, which in turn is a continuation-in-part of U.S.application Ser. No. 136,214, filed Dec. 18, 1987 now abandoned.

FIELD OF THE INVENTION

This invention relates to a method for the preparation of novelcompositions which contain both hydrophobic materials and polymericlatex particles. It also relates to a method for the preparation ofwater-insoluble biological reagents composed of biological compoundsattached to the polymeric latex particles containing hydrophobicmaterials.

BACKGROUND OF THE INVENTION

Several techniques have been described and used to distributehydrophobic compounds, particularly non-polymeric compounds, inpolymeric latex particles. For example, U.S. Pat. No. 4,368,258 (issuedJan. 11, 1983 to Fujiwhara et. al.) describes various techniques forincorporating dye-forming compounds and ultraviolet light absorbers intolatex particles for use in photographic materials. Generally, thetechniques all involve mixing a hydrophobe in solid state, awater-miscible organic solvent and a polymeric latex. The latexpurposely contains large amounts of dispersant (also known as asurfactant or emulsifier).

U.S. Pat. No. 4,199,363 (issued Apr. 22, 1980 to Chen) describes aprocess for "loading" hydrophobes into latex particles whereby thecompounds are dissolved within a water-miscible organic solvent. Theprocess generally comprises gradually adding a "loadable" latex (onedesigned to receive the hydrophobe) to an organic solvent solution ofthe hydrophobe in such a Hanner that the hydrophobe is distributed withthe latex particles predominantly. The resulting loaded latex is used invarious photographic materials. The loadable latex further contains asurfactant for minimizing coagulation of latex particles.

While U.S. Pat. No. 4,199,363 provides useful materials for use invarious photographic materials, there is a need for incorporatinghydrophobes in polymeric latex particles to which can be attachedbiological compounds for various biological processes and assays. Forexample, it would be useful to have insoluble dyed reagents for use invarious assays, such as agglutination assays, for the determination of aligand of some type. These reagents could include a latex containingdyed particles to which a receptor has been attached. A receptor is abiological or chemical compound which specifically reacts or binds to aligand of interest. Examples of ligand and receptor include avidin andbiotin, and antibodies against corresponding antigens.

In preparing such biological reagents, it his been found that theprocess described in U.S. Pat. No. 4,199,363 has serious drawbacks. Thedescribed process whereby loadable latex is gradually added to thehydrophobe solution is prone to coagulation if the addition process isnot carefully controlled. However, the likelihood of coagulation can beminimized with the use of surfactants which protect the latex particles.

However, in the preparation of insoluble biological reagents containinga hydrophobe and a biological compound attached to the particles, thepresence of surfactant in the latex adversely affects the activity ofmany biological compounds. For example, the presence of surfactant ofteninterferes with the reaction of antibody with its corresponding antigen.Such interference inhibits accurate and sensitive detection of variousligands in important diagnostic and analytical procedures. Yet, thepresence of a surfactant appears to be essential in the practice of themethod of adding hydrophobe to latex particles which is described inU.S. Pat. No. 4,199,363.

It would be desirable to be able to incorporate hydrophobes into latexparticles for use in the preparation of biological reagents without theuse of surfactants.

SUMMARY OF THE INVENTION

The problems noted above have been overcome with a method for preparinga polymeric latex composition comprising:

A. providing a loadable latex comprising a continuous aqueous phase anda dispersed phase comprising loadable polymeric particles prepared fromone or more ethylenically unsaturated polymerizable monomers,

B. providing a solution of a hydrophobe dissolved in one or morewater-miscible organic solvents,

C. heating the loadable latex to a temperature of from about 30° toabout 95° C. and

D. gradually adding the hydrophobe solution to the heated loadable latexwhile retaining the hydrophobe in solution and the polymeric particlesdispersed so that the polymeric particles and hydrophobe are broughtinto intimate association, and the water-miscible solvent is dilutedwith water to reduce the solubility of the hydrophobe in the continuousphase, whereby the equilibrium distribution of the hydrophobe is drivenaway from the continuous phase toward the polymeric particles of thedispersed phase.

Also provided by this invention is a method of preparing awater-insoluble biological reagent containing a detectable hydrophobe,the method comprising:

A. providing a loadable latex comprising a continuous aqueous phase anda dispersed phase comprising loadable polymeric particles prepared fromone or more ethylenically unsaturated polymerizable monomers, at leastone of the monomers having reactive groups which are capable of reactingwith free amino or sulfhydryl groups of a biological compound,

B. providing a solution of a detectable hydrophobe dissolved in one ormore water-miscible organic solvents,

C. heating the loadable latex to a temperature of from about 30° toabout 95° C.

D. gradually adding the hydrophobe solution to the heated loadable latexwhile retaining the hydrophobe in solution and the polymeric particlesdispersed so that the polymeric particles and hydrophobe are broughtinto intimate association, and the water-miscible solvent is dilutedwith water to reduce the solubility of the hydrophobe in the continuousphase, whereby the equilibrium distribution of the hydrophobe is drivenaway from the continuous phase toward the polymeric particles of thedispersed phase to provide a latex of particles having the hydrophobewith the particles, and

E. reacting the reactive groups of the particles with a biologicalcompound having free amino or sulfhydryl groups to form a biologicalreagent, provided that either the loadable latex is provided essentiallyfree of surfactant, or any surfactant present is removed prior to thereacting step E.

The present invention provides a reliable method for preparing loadedlatices. By "loaded" is meant that a hydrophobe (that is, a hydrophobiccompound) has been incorporated into preformed latex particles. Thismethod avoids the likelihood of coagulation of particles prematurely andeffectively incorporates relatively large amounts of hydrophobe, ifdesired, into the particles.

In a preferred embodiment, the method of this invention uses a loadablelatex which is essentially surfactant-free. While it might be expectedthat the lack of a surfactant or other stabilizing material might bedisadvantageous, it has been found that the latices used herein arestable and can be used to advantage in preparing insoluble biologicalreagents containing hydrophobes.

These advantages are achieved by heating the loadable latex to atemperature of from about 30° to about 95° C. followed by the gradualaddition of the hydrophobe which is dissolved in a water-miscibleorganic solvent. This procedure is opposite that described in U.S. Pat.No. 4,199,363 where the hydrophobe solution is added gradually to thesurfactant-containing loadable latex. The novel method of this inventioncan be used to advantage because the loadable latex is heated to 30°-95°C. prior to adding the hydrophobe solution. Coagulation is therebyavoided, and the use of a surfactant can be avoided as well if sodesired.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention provides a means for preparing alatex containing one or more hydrophobic compounds incorporated therein.The resulting "loaded" latex is useful in a variety of productsincluding, but not limited to, photographic, electrophotographic,diagnostic, therapeutic and clinical materials. Preferably, the latex isuseful for preparing water-insoluble reagents having diagnostic or otherbiological and medical uses. Other uses would be apparent to a worker ofordinary skill in the art.

The loaded polymeric latex compositions prepared according to thepractice of this invention are polymeric latices comprising a continuousaqueous phase and a dispersed phase (or discontinuous) phase comprisingone or more hydrophobes (defined below) distributed in at least aportion of loadable polymeric particles prepared from one or moreethylenically unsaturated polymerizable monomers (defined below).

The method of loading the polymeric particles in a latex is generally asfollows:

A loadable latex is provided with suitable agitation to minimizecoagulation. Although it is not essential, if desired, the latex can bepurified prior to use to remove any residual surfactants, initiators,reaction by-products (for example, initiator fragments) and unreactedmonomers. In preferred embodiments, the latex is prepared in the absenceof surfactants, in which case, purification may or may not be necessary.

A solution of one or more hydrophobes in one or more water-miscibleorganic solvents is also provided. If necessary, the solution can becontinuously agitated to maintain solubility of the hydrophobe therein.

The loadable latex is heated to a suitable temperature which isgenerally above the glass transition temperature of the latex polymer soas to enhance molecular motion of the hydrophobe therein during theloading process. The temperature will depend upon the characteristics ofthe polymer as well as the natural affinity of the hydrophobe for it.Generally, the temperature is in the range of from about 30° to about95° C.

Once the loadable latex has been suitably heated, the hydrophobesolution is gradually added thereto while retaining the hydrophobe insolution in the continuous phase and the particles and hydrophobe arebrought into intimate association. The hydrophobe is then free todistribute itself between the continuous and dispersed phases based onits relative solubilities therein. Gradual addition of the hydrophobicsolution is required so that the solvent is gradually diluted with waterthereby reducing the solubility of the hydrophobe in the continuousphase, and shifting the equilibrium distribution of the hydrophobe awayfrom the continuous phase toward the polymeric particles of thedispersed phase. In this manner, a suitable portion of the hydrophobe,becomes dispersed or dissolved in the polymeric particles so that theyare loaded with hydrophobe. Any precipitation of the hydrophobe orcoagulation among polymeric particles is minimized or avoided entirely.

More specifically, the loadable latex is heated to a temperature of fromabout 30° to about 95° C. and preferably to a temperature of from about40° to about 90° C. prior to addition of the hydrophobe solution. Thisheating is normally done under atmospheric pressure, although reducedpressures can be used if desired. It is important that the temperaturebe at or above the glass transition temperature (Tg) of the polymericparticles. In the case of core/shell particles described in more detailbelow, the temperature should be at or above the Tg of the core polymerto facilitate loading of the hydrophobe into the core. The loadablelatex is usually stirred continuously during the heating and loadingsteps in order to minimize coagulation. However, vigorous agitation isto be avoided to minimize coagulation. Heating can be accomplished inany suitable manner including the use of high temperature baths orjackets external to the latex, or heating rods or coils within thelatex.

The hydrophobe solution need not be heated in the practice of thisinvention. It is preferred to use it at ambient temperature. However,modest heating (that is, to less than the boiling point of the organicsolvent) can be carried out if desired.

After heating the loadable latex, the hydrophobe solution (which can beheated or unheated) is gradually added to the latex as mentioned above.The rate is such that coagulation is minimized, but that sufficientorganic solvent is available to lower the Tg of the polymer of theparticles to facilitate hydrophobe dispersion within the particles.Thus, the rate of addition will depend somewhat on the polymer, solventand hydrophobe being used. A worker skilled in the art could readilydetermine a suitable rate with routine experimentation. Generally, therate of addition is in the range of from about 1 to about 50 ml/min,with a rate of from about 2 to about 30 ml/min being preferred.

The proportion of loadable latex to hydrophobe solution is generallymaintained within the volume ratio of from about 30:1 to about 1:1, andpreferably from about 15:1 to about 1.5:1. The weight ratio ofhydrophobe in the hydrophobe solution to polymeric particles in thelatex is generally from about 1:30 to about 1:1, and preferably fromabout 1:15 to about 1:4.

Optionally, additional water can be added to the loadable latexsimultaneously or subsequent to addition of the hydrophobe solution.This water can be at the same or different temperature as the loadablelatex. This additional dilution of the continuous phase with water maybe useful to further reduce the likelihood of coagulation.

Other optional steps can be followed if desired, such as purificationsteps to remove unwanted materials (such as surfactant and residualmonomers) from the loadable latex prior to use, or to remove residualsolvent, surfactant and hydrophobe after the loading process. Theequipment and procedures needed for such steps are well known to oneskilled in the art. If the loaded latex is to be used to prepare abiological reagent (as described herein), any residual surfactant mustbe removed prior to attaching biological compounds to the latexparticles.

The aqueous loadable latices which are useful as starting materials inthe practice of this invention comprise an aqueous continuous phase andloadable polymeric particles as a dispersed phase. The latices arepreferably essentially free of surfactant or other colloidal orpolymeric dispersing agents which are generally used to keep laticesfrom coagulation.

The loadable polymeric particles useful in this invention can be chosenfrom among those which meet the following test:

At 25° C. the loadable polymeric particles being tested must (a) becapable of forming a latex with water at a particle concentration offrom about 0.2 to about 20 percent, based on total latex weight, and (b)when a 100 ml sample of the latex is then mixed with an equal volume ofthe water-miscible organic solvent to be employed in forming the loadedpolymeric latex composition desired, stirred and allowed to stand for 10minutes, it exhibits no observable coagulation of polymeric particles.

It will be appreciated that the loadable particles can comprise of avariety of different loadable polymers. The particles can behomogeneous, meaning they are composed of the same polymer throughout,or they can be composed of two or more polymers, such as core/shellpolymer particles, graft copolymers, and other embodiments readilyapparent to one skilled in the art. Preferably, the particles arecore/shell polymers which are described in more detail below.

The particles are composed of polymers which are prepared from one ormore ethylenically unsaturated polymerizable monomers, of which hundredsare known, and others are readily prepared by a skilled worker in theart. Generally, the monomers include, but are not limited to, monomersof the following groups:

(i) Ethenic monomers of the formula:

    H.sub.2 C═CRR.sup.1

wherein R is hydrogen, halo or vinyl, and R¹ is hydrogen, halo,substituted or unsubstituted lower alkyl (for example, methyl or ethyl),or cyano if R is hydrogen. Examples of such monomers include isoprene,chloroprene, 1,3-butadiene, propenenitrile, vinylidene chloride, vinylchloride, vinyl fluoride, ethylene, propylene and acrylonitrile.

(ii) Vinyl monomers of the formula:

    CHR.sup.3 ═CR.sup.4 R.sup.5

wherein R³ and R⁴ are independently hydrogen or substituted orunsubstituted lower alkyl (for example, methyl, chloromethyl or ethyl)and R⁵ is a substituted or unsubstituted aryl group (generally of 6 to14 carbon atoms in the aromatic ring, for example phenyl, tolyl, xylylor naphthyl), a substituted or unsubstituted cycloalkyl (generally of 5to 8 carbon atoms, for example, cyclopentyl, cyclohexyl,4-methylcyclohexyl or cyclooctyl) or a substituted or unsubstitutedheterocyclic group (generally of 5 to 7 carbon and heteroatoms in thering, for example imidazolyl, pyrrolidonyl or pyridyl). Representativemonomers of this formula include styrene, o-vinyltoluene,p-vinyltoluene, p-chlorostyrene, p-bloromethylstyrene,m-chloromethylstyrene, α-Methyletyrene, 2,5-dimethylstyrene,2-ethylstyrene, 2-vinylmesitylene, 1-vinylnaphthalene, 1-vinylimidazole,4-vinylpyridine and t-butyletyrene.

(iii)Monomers which are 2-alkenoic acids or esters or anhydrides thereofwhich are generally of the formula:

    HR.sup.6 C═CR.sup.7 COOR.sup.8

wherein R⁶ is hydrogen, --COOR⁸ or substituted or unsubstituted loweralkyl (for example, methyl, carboxymethyl, chloromethyl, ethyl, propylor t-butyl) and R⁷ is hydrogen, halo or substituted or unsubstitutedlower alkyl as described above. R⁸ is hydrogen, substituted orunsubstituted alkyl or haloalkyl of 1 to 20 carbon atoms. Representativemonomers of this formula include acrylic acid, methacrylic acid, butylacrylate, methyl methacrylate, ethyl methacrylate, 2-ethylhexylmethacrylate and 2-acetoacetoxyethyl methacrylate.

(iv) Crosslinkable monomers, for example, those having two or moreethylenically unsaturated polymerizable groups, or those which arehardenable. Representative monomers are described in ResearchDisclosure, publication 19551, July, 1980, page 304 and includedivinylbenzene, ethylene dimethacrylate, N,N'methylenebisacrylamide,2,2-dimethyl-1,3-propylene diacrylate, allyl acrylate, ethylidynetrimethacrylate and ethylene diacrylate.

(v) Ionic monomers having one or more anionic or cationic moieties, suchas sulfates, sulfonates, phosphonates, carboxyl, quaternary amoniumgroups and others known in the art. Representative monomers includesodium 2-acrylamido-2-methylpropanesulfonate, 2-carboxyethyl acrylate,styrenesulfonic acid, potassium salt and others known to one skilled inthe art.

(vi) Monomers having requisite reactive groups which will directly orindirectly react with free amine or sulfhydryl groups of a biologicalcompound such as an immunological compound, protein, enzyme or othercompound of interest. Representative of such monomers include, but arenot limited to, monomers having an active halogen atom (such as vinylchloroacetate, chloroalkylated vinyl aromatics, for examplechloromethylstyrene, or chloroalkyl acrylic or methacrylic esters, forexample chloroethyl methacrylate, 3-chloro-2-hydroxypropyl methacrylate,and 3-chloropropyl acrylate), monomers having one or more pendantcarboxyl groups or their functional equivalents (such as acrylic acid,methacrylic acid, itaconic acid, maleic acid or their anhydrides),monomers containing epoxy groups (such as glycidyl acrylate, glycidylmethacrylate, vinyl glycidyl ether or methallyl glycidyl ether),monomers containing isocyanate groups (such as isocyanatoethyl acrylate,isocyanatoethyl methacrylate, or α, α-dimethylmetaisopropenylbenzylisocyanate), amine-containing monomers [such as 2-aminoethylmethacrylate, and N-(3-aminopropyl)methacrylamide], monomers containingan aziridine group [such as vinylcarbamoyl aziridine, acryloylaziridine, methacryloyl aziridine, N-acryloylaziridine and2-(1-aziridinyl)ethyl acrylate], monomers containing aldehyde groups(such as vinylbenzaldehyde or acrolein), 2-substituted ethylcarbonylcontaining monomers (such as 2-chloroethyl acrylate, 2-chloroethylmethacrylate, 2-methyloulfonyloxyethyl methacrylate and2-@tolylsulfonyloxyethyl acrylate) or monomers having pendant activated2-substituted ethyloulfonyl or vinyloulfonyl groups [such as thosedescribed in U.S. Pat. Nos. 4,161,407 (issued Jul. 17, 1979 to Campbell)and 4,548,870 (issued Oct. 22, 1985 to Ogawa et. al.)] and others knownto one skilled in the art.

Preferred monomers which can be used to prepare polymers useful in thepractice of this invention include those having active halomethyl groupsof 1 to 3 carbon atoms and the activated 2-substituted ethylsulfonyl andvinylsulfonyl monomers.

Preferred monomers having active halomethyl groups of 1 to 3 carbonatoms include chloromethylstyrene and bromomethylstyrene.

Preferred activated 2-substituted ethyloulfonyl and vinylsulfonylmonomers can be represented by the formula: ##STR1## wherein R' ishydrogen or substituted or unsubstituted alkyl (generally of 1 to 6carbon atoms, such as methyl, ethyl, isopropyl or hexyl. Preferably, R'is hydrogen or methyl.

R'' is --CH═CHR''' or --CH₂ CH₂ X wherein X is a leaving group which isdisplaced by a nucleophile or is eliminated in the form of EX bytreatment with a base (such as halo, acetoxy, alkyloulfonyloxy such asmethyloulfonyloxy, arylsulfonyloxy such as p-tolylsulfonyloxy,trialkylammonio, for example, a trimethylammonio salt or pyridiniosalt). R''' is hydrogen, substituted or unsubstituted alkyl (generallyof 1 to 6 carbon atoms as defined for R'), or substituted orunsubstituted aryl (generally of 6 to 12 nuclear carbon atoms, such asphenyl, naphthyl, xylyl or tolyl). Preferably, R'' is --CH₂ CH₂ X. Thisgroup, which is an activated 2-substituted ethyl group, can besubstituted with any group which does not impair the displacement of theleaving group X.

L is a linking group which can be a substituted or unsubstitutedalkylene generally having 1 to 20 carbon and hetero atoms in thebackbone. This definition of alkylene is meant to include alkylenegroups interrupted or terminated with oxy, thio, --NR⁹ --[wherein R⁹ ishydrogen, substituted or unsubstituted alkyl of 1 to 6 carbon atoms(such as methyl, chloromethyl or 2-hydroxyethyl) or substituted orunsubstituted aryl of 6 to 10 carbon atoms (such as phenyl, naphthyl orxylyl)], ester (--COO--), amide (--CONH--), ureylene ##STR2## sulfonyl(--SO₂ --), carbonate, sulfonamide, azo, phosphono or other similargroups. Representative alkylene groups include methylene, ethylene,isobutylene, hexamethylene, carbonyloxyethoxycarbonyl,methylenebis(iminocarbonyl), carbonyloxydodecylenecarbonyloxyethylene,carbonyliminomethyleneiminocarbonyliminoethylene,carbonyliminomethyleneiminocarbonylethylene and other groups describedor suggested by U.S. Pat. Nos. 4,161,407 and 4,548,870, noted above.

L can also be substituted or unsubstituted arylene generally having 6 to12 nuclear carbon atoms. Representative arylene groups includephenylene, tolylene, naphthylene and others noted in the patentsmentioned above. Also included in this definition of L are divalentgroups which are combinations of one or more of each of the alkylene andarylene groups defined above (for example, arylenealkylene,alkylenearylenealkylene and others readily determined by one of ordinaryskill in the art). Preferably, L is substituted or unsubstitutedphenylenealkylene, phenylenealkylene substituted with one or more alkylgroups (as defined for R'), alkoxy groups (generally of 1 to 6 carbonatoms, for example, methoxy, propoxy or butoxy) or halo groups, orcarbonyliminomethyleneiminocarbonylethylene.

Representative useful monomers include m andp-(2-chloroethylsulfonylmethyl)styrene, m andp-[2-(p-tolylsulfonyloxy)ethylsulfonylmethyl]styrene, m andp-vinylsulfonylmethylstyrene, N-[m andp-(2-chloroethylsulfonylmethyl)phenyl]acrylamide, andN-[2-(2-chloroethylsulfonyl)ethylformamidomethyl]acrylamide. acrylamide.The first monomer is preferred.

The polymeric particles used in the practice of this invention arewater-insoluble latex particles having a particle size greater thanabout 0.01 μmeters, preferably in the range of from about 0.01 to about5 μmeters, and more preferably from about 0.1 to about 3 μmeters. Theparticles are present in the loadable latex in an amount of from about0.2 to about 30 percent based on total latex weight. Preferably, theamount is from about 0.5 to about 15 weight percent.

Preferred particles are composed of at least two separate polymers, forexample as a core/shell polymer or a graft copolymer. Core/shellpolymers are particularly useful where the core polymer has a higheraffinity for a loaded hydrophobe in comparison to the shell polymer. Inaddition, the shell polymer is particularly useful when it is preparedfrom Lo monomers having the reactive groups described above.

In general, useful core polymers have a glass transition temperature(identified herein as Tg₁) less than about 100° C. and preferably fromabout -25° to about 95° C. in order to facilitate solubilization andimmobilization of the hydrophobe in the core polymer. This Tg₁ is acalculated value determined using the following equation [with the glasstransition temperature values ° K. (Kelvin) which can be readilyconverted to ° C.]: ##EQU1## wherein m1, m2, . . . mn represent theindividual monomers from which the first polymer is derived and identifythe Tg (° K.) of the homopolymer prepared from each individual monomer,X₁, X₂, . . . X_(n) represent the weight fractions of the monomers usedto prepare the first polymer, and n represents the number of monomersused to prepare the first polymer.

Representative polymers of which the core of the particles can becomposed include the following materials (the Tg₁ values have beencalculated for some of the polymers) : poly(styrene-co-2-acetoacetoxyethyl methacrylate) (50:50, 70:30, 85:15 and 95:5 molarratios, having Tg₁ s of 27°, 47°, 69° and 91° C. respectively),poly(styrene-co-m and p-chloromethylstyrene-co-2-hydroxyethyl acrylate)67:30:3 molar ratio), poly(styrene-co-n-butyl acrylate) (78.7:21.3 molarratio, Tg₁ of 47° C.) and poly-(styrene-co-benzyl acrylate) (90:10 molarratio).

The shell of the particles comprises a second polymer which providesreactive sites for covalent bonding of the biological compound, andsufficient swellability in water-miscible organic solvents used in theloading of hydrophobe into the core of the particles without encouragingretention of the hydrophobe in the shell.

Generally, the second polymer has a Tg₂ which is equal to or greaterthan the term [Tg₁ less 10° C.]. Tg₂ is usually greater than Tg₁, but itcan be equal to Tg₁, or as much as 10° C. less than Tg₁.

Representative polymers of which the shell of the particles can becomposed include the following (the Tg₂ values have been determined forsome of the materials): poly(m and p-chloromethylstyrene) (Tg₂ of 82°C.), poly(styrene-co-m and p-chloromethylstyrene-co-2-hydroxyethylacrylate (67:30:3 molar ratio), poly(styrene-co-m andp-chloroethyloulfonylmethylstyrene) (95.5:4.5 molar ratio, Tg₂ of 105°C.), poly(styrene-co-N-[m andP-(2-chloroethylsulfonylmethyl)phenyl]acrylamide} (99.3:0.7 molarratio), poly(m and p-chloromethyletyrene-co-methacrylic acid)(95:5, 98:2and 99.8:0.2 molar ratio, Tg₂ s of 85°, 83° and 82° C., respectively),poly(styrene-co-m and p-chloroethylsulfonylmethyletyrene-co-methacrylicacid)(93.5:4.5:2 molar ratio), poly(styrene-p-N-[m andp-(2-chloroethylsulfonylmethyl)phenyl]acrylamide-co-methacrylicacid}(97.3:0.7:2 molar ratio), poly-(styrene-co-m andp-vinylbenzaldehyde)(95:5 molar ratio), poly(styrene-co-m andp-vinylbenzaldehyde-co-methacrylic acid)(93:5:.2 molar ratio),poly(styrene-co-m and p-chloromethylstyrene)(70:30 molar ratio, Tg₂ of96° C.) and poly(styrene-co-methacrylic acid)(90:10 molar ratio, Tg₂ of113° C.).

The polymeric particles can be prepared using any suitablepolymerization technique, including emulsion (including batch,semi-continuous and continuous) and suspension polymerizationtechniques, graft copolymerization, and others known to one skilled inthe polymer chemistry art. Emulsion polymerization is preferred as itcan be used to provide particles without the use of surfactants oremulsifiers as described for example in U.S. Pat. No. 4,415,700 (issuedNov. 15, 1983 to Btaz et. al.) and Research Disclosure publication 15963(July, 1977). Research Disclosure is a publication available fromKenneth Mason Publications, Ltd., The Old Harbourmaster's, 8 NorthStreet, Emsworth, Hampshire P010 7DD, England. Continuous emulsionpolymerization is the most preferred technique, as described in thenoted Research Disclosure publication. Other details of preparatorymethods can be found in U.S. Pat. Nos. 4,161,407 and 4,548,870, notedabove.

More particularly, staged emulsion polymerization can be used to providecore/shell polymers composed of two different polymers. Emulsionpolymerization of the core is carried to substantial completion bycontinuously adding reactants to a reaction vessel under standardconditions. Monomers and initiators needed to make the shell polymer arethen continuously added to the vessel containing the latex of the corepolymer. In this manner, the shell has a definite known compositionrather than being a mixture of core and shell monomers. Representativedetails of preparing the core-shell polymeric particles useful in thisinvention are provided in the Example below.

The core polymer of the particle generally comprises from about 30 toabout 80, and preferably from about 40 to about 70, weight percent ofthe particle.

Further details of the preferred core/shell polymer particles can befound in copending and commonly assigned U.S. application Ser. No.98,583, filed Sep. 18, 1987 by Sutton et. al.

To be considered a hydrophobe as that term is used herein, the compoundmust be essentially insoluble in distilled water at 25° C. Preferably,the dissolved concentration of hydrophobe in water under thoseconditions should be less than about 0.5 percent by weight at 25° C.based on the weight of the water. Any such hydrophobe can be employed inthe practice of this invention which is or can be dissolved in a liquidconsisting of one or more water-miscible organic solvents. Preferably,the hydrophobe must be soluble in the liquid at a concentration of atleast about 0.2 percent based on the total weight of the resultingsolution.

Although the practice of the present invention is not limited toparticular hydrophobes, various classes of compounds are particularlyuseful. These classes include various hydrophobic photographic addenda,insecticides, herbicides, miticides, rodenticides, vitamins, enzymes,hormones and others known in the art. for example, those described inU.S. Pat. No. 4,199,363 (noted above) and the references mentionedtherein.

Particularly useful hydrophobes are those normally considered tracermaterials in biological diagnostic and analytical procedures, such asradiolabeled compounds which emit gamma rays, bioluminescent compounds,chemiluminescent compounds, chromogens such as water-insoluble dyes anddye-formers (including leuco dyes, for example imidazoles), fluorescentcompounds or dyes, for example rare earth chelates.

In a preferred embodiment of this invention, the tracer material is anaromatic dye or leuco dye which is solubilized in the core polymer ofcore/shell particles. Such dyes or dye-providing materials can be of anysuitable color which is readily observed in an assay. Generally, theycontain one or more aromatic or other moieties which render them solublein organic solvents which are water-miscible. Such dyes are notconsidered water-soluble. Preferred aromatic dyes include azo dyes, suchas Oil Red EGN (available from Aldrich Chemical Co.) and Kodak Oil Red 0(available from Eastman Kodak Co.). Other useful dyes would be readilydetermined by a skilled chemist with routine experimentation by seeingwhich dyes have the desired organic solvent solubility and selectivesolubility in a particular core polymer. Particularly useful leuco dyesinclude the triarylimidazoles described, for example in U.S. Pat. No.4,089,747 (issued May 16, 1978 to Bruschi) such as2-(4-hydroxy-3,5-dimetboxyphenyl)-4,5-bis(4-methoxyphenyl)imidazolewhich provides a red dye upon reduction.

The amount of hydrophobe in the particles will depend upon thehydrophobe, polymer and intended use of the resulting loaded latex.Generally, for the core/shell particles, the amount is generally in therange of from about 0.1 to about 15 percent, based on particle weight.

The water-miscible organic solvents useful in the practice of thisinvention are generally those which:

(a) can be dissolved in (that is, they are miscible in) distilled waterat 20° C. to the extent of at least about 20 parts by volume of solventin 80 parts by volume of water,

(b) have boiling points (at atmospheric pressure) above about -10° C.

(c) do not detrimentally react chemically with aqueous laticescontaining the loadable polymer particles which are useful in thepractice of this invention, and

(d) do not dissolve more than about 5 weight percent of such loadablepolymer particles at 20° C.

Representative useful solvents include, but are not limited to, alcohols(for example, methanol, ethanol, isopropanol, ketones (for example,acetone, methyl ethyl ketone), amides (for example, dimethylformamide),nitriles (for example, acetonitrile), tetrahydrofuran,N-methyl-2-pyrrolidone, dimethyl sulfoxide and mixtures thereof. Ofthese, acetone, methanol and acetonitrile are preferred when thehydrophobe is soluble therein.

The amount of hydrophobe present in the hydrophobe solution can varydepending upon the hydrophobe, solvents and loadable latex used in themethod. Generally, the hydrophobe is present in an amount of from about0.05 to about 5 percent based on total solution weight.

The preparation of loaded latices is described in general terms above,and an example of a preferred procedure is provided in Example 1 below.

Once a loaded latex has been prepared, a biological compound of interestcan be attached to the particles, assuming, of course, that theparticles have surface reactive groups which are capable of reactingwith the free amine or sulfhydryl groups of the compound.

The general procedure for preparing a biological reagent includescovalently attaching the biological compound of interest to theparticles using generally known reactions. With many pendant groups, forexample the haloalkyl, 2-substituted activated ethyloulfonyl andvinylsulfonyl, the compound can be directly attached to the particles.Generally, the polymer particles are mixed with the compound in anaqueous buffered solution (pH generally from about 6 to about 10) and aconcentration of from about 0.01 to about 40 weight percent polymerparticles (preferably from about 0.01 to about 10 weight percent). Theamount of compound is at a ratio of compound to polymer of from about0.1:1000 to about 1:10, and preferably from about 1:100 to about 1:10.Mixing is carried out at a temperature in the range of from about 5 toabout 50° C., and preferably at from about 5 to about 40° C. for fromabout 0.5 to about 48 hours. Any suitable buffer can be used. Thedetails of a representative preparatory procedure are illustrated inExample 2 below.

In some instances, the pendant reactive groups on the outer surface mustbe modified or activated in order to cause covalent attachment of thebiological compound. For example, carboxyl groups must be activatedusing known carbodiimide chemistry, or using the carbamoyloniumchemistry described in copending and commonly owned U.S. applicationSer. No. 98,429 entitled "Attachment of Compounds to Polymeric ParticlesUsing Carbamoylonium Compounds" filed on Sep. 18, 1987 by Sutton et. al.

In other instances, an epoxy group on the outer surface can behydrolyzed to form a diol compound capable of reacting with cyanogenbromide which can act as a coupling agent for amine groups in thebiological compound. Aldehydes can react directly with amines to form aShiff's base which can be subsequently reduced to form a covalent link.Alternatively, the aldehyde can be oxidized to an acid and chemistryidentified above for carboxyl groups can be used to form an amidelinkage.

The conditions of the reaction procedures described above are well knownin the art and would require merely routine experimentation forimplementing.

The following examples illustrate the practice of the present invention,but it is not to be construed as so limited.

EXAMPLE 1 Preparation of Loaded Latex

This example illustrates the preparation of a loadable latex ofcore/shell polymer particles, and the loading of a hydrophobe into it.

Preparation of Loadable Latex

The three solutions outlined below were continuously added to a 1300 mlvessel containing deoxygenated water at 80° C. at the indicated rates:

Solution 1: Styrene (103 g), 2-acetoacetoxyethyl methacrylate (91 g) and1-dodecanethiol (1.9 g) at 1.08 g/min. for 180 minutes.

Solution 2: Ammonium peroulfate (6.5 g) and distilled, deoxygenatedwater (651 g) at 2.13 g/min. for 300 minutes.

Solution 3: Sodium pyrosulfite (3.24 g) and distilled water (651 g) at2.17 g/min. for 300 minutes.

After 180 minutes, Solution 1 was exhausted, and replaced with asolution of M and p-chloromethylstyrene (130 g) and 1-dodecanethiol (1.3g) which was added at a rate of 1.08 g/min. for 120 minutes. The finalreactor contents were 11.45% solids. After five days of dialysis, thelatex was 8.7% solids, and the average size of the resulting core/shellparticles was about 0.34 PM as measured by transmission electronmicroscopy.

Loading Hydrophobe into the Particles

Kodak Oil Red 0 Dye (available from Eastman Kodak Co.) (2.5 g) wasdissolved in acetonitrile (150 g). To a 60 g sample of the dialyzedloadable latex described above was added distilled water (290 g), andthe resulting mixture was heated to 70° C. with stirring. To the hotlatex solution were added 30 g portions of the dye solution, one portionevery 30 minutes, until all had been added. The resulting dispersion wasfiltered, stripped of residual acetonitrile under reduced pressure, andrefiltered to yield 70 g of a 4.82% mixture of nonagglutinatedcore/shell particles having dye in the cores only. The dye content wasdetermined spectrophotometrically to be 8.9% (based on polymer weight).

EXAMPLE 2 Preparation of a Biological Reagent

This example illustrates the practice of the present invention toprepare a biological reagent useful for the determination ofStreptococcus A antigen.

Monoclonal antibodies to Streptococcus A antigen and casein werecovalently immobilized on the particles of the loaded latex described inExample 1 as follows: to 0.6 ml of 50 remolar borate buffer (pH 8.5) wasadded 0.1 mg of total protein comprised of a 10:1 mixture of anti-StrepA antibody (2.9 mg/ml solution in phosphate buffered saline solution,known in the art as PBS) and casein (10 mg/ml water). After mixing, 41.5μl of a 5% suspension of the polymeric latex particles were added (toprovide 0.3% solids) and the resulting solution was rotated(end-overend) for 24 hours at 37° C. to effect covalent attachment ofthe antibody and the casein to the particles to form a biologicalreagent.

EXAMPLE 3 Preparation of hCG Biological Reagent

This example illustrates the preparation of a biological reagent usefulfor the determination of human chorionic gonadotropin (hCG).

Core/shell polymeric particles of a loadable latex were imbibed with OilRed EGN dye according to the procedure shown in Example 1 above. Theparticle cores were composed of poly(styrene-co-2-acetoacetoxyethylmethacrylate) (85:15 molar ratio), and the particle shells were composedof poly(m and p-chloromethylstyrene ethacrylic acid) (99.8:0.2 molarratio). The particles had an average diameter of about 0.32 micrometer.

Monoclonal antibodies to two different epitopic sites of hCG werecovalently immobilized on these particles as follows: to 0.6 ml of 50mmolar borate buffer (pH 8.5) were added 0.1 2g of 10:1 mixture of hCGantibody (2.9 mg/ml phosphate buffered saline solution) and casein (10mg/ml water). After mixing, 41.5 pl of a 5% suspension of the latexparticles described above were added and the resulting suspension wasrotated (end-overend) for 24 hours at 37° C. to effect covalentattachment of the antibodies and casein to the particles to form abiological reagent.

EXAMPLE 4 Loading Latex of Poly(styrene-co-m and p-chloromethylstyrene)Particles

This example illustrates the practice of this invention by loading thecommercially available hydrophobe, KODAK Oil Red 0 dye, into a loadablelatex comprising latex particles (0.42 μm average size) ofpoly(styrene-co-m and p-chloromethylstyrene) (77.3:22.7 molar ratio).

This method was carried out by adding water (26 g) to a sample of thelatex (54 g, 1.87% solids). The latex was then heated to 70° C. followedby addition of a hydrophobe solution (20 g) comprising the dye (0.1 g)in acetonitrile at a rate of 5 g/min. After mixing the resultingdispersion for about one hour, it was filtered through a coarse filterand the acetonitrile was removed under reduced pressure. The loadedlatex was then filtered through Reeves Angel 230 filter paper, yielding54 g of loaded latex of dyed particles (1% solids).

EXAMPLE 5 Loading Latex of Poly(styrene-co-m andp-chloromethylstyrene-co-2-hydroxyethylacrylate)

This example is similar to Example 4 except that the loadable latexcomprised particles (0.77 μm average size) of poly(styrene-co-m andp-chloromethylstyrene-co-2-hydroxyethyl acrylate)(67:30:3 molar ratio).

To a sample of the latex (22 g, 2.2% solids), was added 68 ml of water.The latex was then heated to 80° C., and a hydrophobe solution (30 g)comprising KODAK Oil Red 0 dye (0.25 g) in acetonitrile was graduallyadded (6 g/min.) with mixing. The resulting dispersion was purified asdescribed in Example 4, yielding 24 g of a loaded latex of dyedparticles (0.64% solids, 9.2% dye by weight of particles).

EXAMPLE 6 Loading Leuco Dye into Latex Particles

This example illustrates the practice of the present invention toincorporate a hydrophobic leuco dye into the core of core/shell polymerparticles.

A latex (12.25 g, 10.2 % solids) containing core/shell polymer particleshaving a core of poly(styrene-co-2-acetoacetoxyethyl methacrylate)(70:30 mole ratio) and a shell of poly[styrene-co-m andp-(2-chloroethyleulfonylmethyl)styrene (60:40 molarratio)-co-methacrylic acid](94.5:4.5:1 molar ratio) was diluted withdistilled water (71.09 ml). The particles had an average diameter of1.50 pm. After heating to about 30° C. and while being moderatelystirred (that is, without vigorous agitation), a leuco dye solution(0.56 g) was added to the latex.

This leuco dye solution consisted of2-(4-hydroxy-2,3-dimethoxyphenyl)-4,5-bis-(4-methoxyphe nyl)imidazole(1.66 g) and sodium meta bisulfate (0.1 g) dissolved in ethanol (100 ml)(2.24 % solids). Additional sodium meta bisulfite (0.05 g) was thenadded to the resulting latex mixture. After moderate stirring for 15minutes, the mixture was filtered through a milk filter. The resultinglatex containing leuco dye "loaded" particles was obtained at 1.6 %solids.

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

I claim:
 1. A method of preparing a water-insoluble biological reagentcontaining a detectable hydrophobe, said method comprising:A. providinga stable loadable latex comprising a continuous aqueous phase and adispersed phase comprising loadable polymeric particles having anaverage diameter of from about 0.01 having an average diameter of fromabout 0.01 to about 5 micrometers, said particles being prepared fromone or more ethylenically unsaturated polymerizable monomers, at leastone of said monomers having reactive groups which are capable ofreacting with free amino or sulfhydryl groups of a biological compound,B. providing a solution of a detectable hydrophobe dissolved in one ormore water-miscible organic solvents, said hydrophobe being soluble indistilled water in an amount of less than about 0.5 weight percent at25° C. and being soluble in a water-miscible organic solvent in anamount of at least about 0.2 weight percent, and said hydrophobe beingselected from the group consisting of radio-labeled compounds,bioluminescent compounds, chemiluminescent compounds, chromogens,fluorescent compounds or dyes, photographic addenda, insecticides,herbicides, miticides, rodenticides, enzymes, hormones and vitamins. C.heating said loadable latex to a temperature of from about 30° to about95° C., D. gradually adding said hydrophobe solution to said heatedloadable latex while retaining said hydrophobe in solution and saidpolymeric particles dispersed so that said polymeric particles andhydrophobe are brought into intimate association, and saidwater-miscible solvent is diluted with water to reduce the solubility ofsaid hydrophobe in said continuous phase, whereby the equilibriumdistribution of said hydrophobe is driven away from said continuousphase toward said polymeric particles of said dispersed phase to providea latex of particles having said hydrophobe within the particles, saidaddition being accomplished with continuous stirring, but withoutvigorous agitation, of said loadable latex, and E. reacting the reactivegroups of said particles with a biological compound having free amino orsulfhydryl groups to form a biological reagent, provided the either saidloadable latex is provided free of surfactant and colloidal or polymericdispersing agents, or any surfactant and dispersing agent present isremoved prior to said reacting step E.
 2. The method of claim 1 whereinsaid biological compound is a protein.
 3. The method of claim 2 whereinsaid protein is an antibody.
 4. The method of claim 3 wherein saidprotein is an antibody directed to either Streptococcus A of humanchorionic gonadotropin.
 5. The method of claim 1 wherein saidwater-miscible organic solvent is selected from the group consisting ofalcohols, ketones, amides, nitriles, tetrahydrofuran,N-methyl-2-pyrrolidone, dimethyl sulfoxide and mixtures thereof.
 6. Themethod of claim 1 wherein said loadable polymeric particles arecore-shell polymeric particles prepared from first and second polymersfor said core and shell, respectively, said second polymer beingprepared from at least one monomer which has said reactive groups forreaction with said biological compound.
 7. The method of claim 1 whereinsaid hydrophobe is a dye or leuco dye.
 8. The method of claim 1 whereinsaid reactive groups are halomethyl, vinylsulfonyl, carboxy or activated2-substituted ethylsulfonyl groups.
 9. The method of claim 1 whereinsaid hydrophobe is present in said hydrophobe solution is an amount offrom about 0.05 to about 5 percent based on total solution weight.